Hormonal and synaptic influences of serotonin on adult neurogenesis.

New neurons are incorporated into the adult brains of a variety of organisms, from humans and higher vertebrates, to non-vertebrates such as crustaceans. In virtually all of these systems serotonergic pathways appear to provide important regulatory influences over the machinery producing the new neurons. We have developed an in vitro preparation where adult neurogenesis can be maintained under highly controlled conditions, and are using this to test the influence of hormones on the production of neurons in the crustacean (Homarus americanus) brain. Serotonin levels have been manipulated in this in vitro preparation, and the resulting effects on the rate of neurogenesis have been documented. In addition we have compared in vitro influences of serotonin with results acquired from in vivo exposure of whole animals to serotonin. These experiments suggest that there are multiple mechanisms and pathways by which serotonin may regulate neurogenesis in the crustacean brain: (1) serotonin is effective in regulating neurogenesis at levels as low as 10(-10)M, suggesting that circulating serotonin may have hormonal influences on neuronal precursor cells residing in a vascular niche or the proliferation zones; (2) contrasting effects of serotonin on neurogenesis (up- vs. down-regulation) at high concentrations (10(-4)M), dependent upon whether eyestalk tissue is present or absent, indicate that serotonin elicits the release of substances from the sinus glands that are capable of suppressing neurogenesis; (3) previously demonstrated (Beltz, B.S., Benton, J.L., Sullivan, J.M., 2001. Transient uptake of serotonin by newborn olfactory projection neurons. Proc. Natl. Acad. Sci. USA 98, 12730-12735) serotonergic fibers from the dorsal giant neuron project directly into the proliferation zone in Cluster 10, suggest synaptic or local influences on neurogenesis in the proliferation zones where the final cell divisions and neuronal differentiation occur. Serotonin therefore regulates neurogenesis by multiple pathways, and the specific mode of influence is concentration-dependent.

Nitric oxide in the crustacean brain: regulation of neurogenesis and morphogenesis in the developing olfactory pathway.

Nitric oxide (NO) plays major roles during development and in adult organisms. We examined the temporal and spatial patterns of nitric oxide synthase (NOS) appearance in the embryonic lobster brain to localize sources of NO activity; potential NO targets were identified by defining the distribution of NO-induced cGMP. Staining patterns are compared with NOS and cyclic 3,5 guanosine monophosphate (cGMP) distribution in adult lobster brains. Manipulation of NO levels influences olfactory glomerular formation and stabilization, as well as levels of neurogenesis among the olfactory projection neurons. In the first 2 days following ablation of the lateral antennular flagella in juvenile lobsters, a wave of increased NOS immunoreactivity and a reduction in neurogenesis occur. These studies implicate nitric oxide as a developmental architect and also support a role for this molecule in the neural response to injury in the olfactory pathway.

Regulation of serotonin levels by multiple light-entrainable endogenous rhythms.

This study examined whether serotonin levels in the brain of the American lobster, Homarus americanus, are under circadian control. Using high-performance liquid chromatography and semi-quantitative immunocytochemical methods, we measured serotonin levels in the brains of lobsters at six time points during a 24-h period. Lobsters were maintained for 2 weeks on a 12 h:12 h light:dark cycle followed by 3 days of constant darkness. Under these conditions, brain serotonin levels varied rhythmically, with a peak before subjective dusk and a trough before subjective dawn. This persistent circadian rhythm in constant darkness indicates that serotonin levels are controlled by an endogenous clock. Animals exposed to a shifted light cycle for >10 days, followed by 3 days in constant darkness, demonstrate that this rhythm is light entrainable. Separate analyses of two pairs of large deutocerebral neuropils, the accessory and olfactory lobes, show that serotonin levels in these functionally distinct areas also exhibit circadian rhythms but that these rhythms are out of phase with one another. The olfactory and accessory lobe rhythms are also endogenous and light entrainable, suggesting the presence of multiple clock mechanisms regulating serotonin levels in different brain regions.

Transient uptake of serotonin by newborn olfactory projection neurons.

A life-long turnover of sensory and interneuronal populations has been documented in the olfactory pathways of both vertebrates and invertebrates, creating a situation where the axons of new afferent and interneuronal populations must insert into a highly specialized glomerular neuropil. A dense serotonergic innervation of the primary olfactory processing areas where these neurons synapse also is a consistent feature across species. Prior studies in lobsters have shown that serotonin promotes the branching of olfactory projection neurons. This paper presents evidence that serotonin also regulates the proliferation and survival of projection neurons in lobsters, and that the serotonergic effects are associated with a transient uptake of serotonin into newborn neurons.

Effects of serotonin depletion on local interneurons in the developing olfactory pathway of lobsters.

During embryonic life, the growth of the olfactory and accessory lobes of the lobster brain is retarded by serotonin depletion using 5,7-dihydroxytryptamine (5,7-DHT) (Benton et al., 1997). The local and projection interneurons that synapse with chemosensory cells in the olfactory lobes are potential targets of this depletion. This study documents proliferation and survival in the local interneuron cell clusters, and examines the differentiation of a prominent local interneuron, the serotonergic dorsal giant neuron (DGN), following serotonin depletion. An increase in dye coupling between the DGN and nearby cells is seen after serotonin depletion. However, morphometric analyses of individual DGNs in normal, sham-injected, and 5,7-DHT-treated embryos show that the general morphology and size of the DGNs are not significantly altered by serotonin depletion. Thus, the DGN axonal arbor occupies a greater proportion of the reduced olfactory lobes in the 5,7-DHT-treated embryos than in normal and sham-injected groups. The paired olfactory globular tract neutrophils (OGTNs), where olfactory interneurons synapse onto the DGNs, are 75% smaller in volume than the comparable region in either sham-injected or normal embryos. In vivo experiments using bromodeoxyuridine (BrdU) show that proliferation in the local interneuron soma clusters is reduced by 5,7-DHT treatment and that survival of newly proliferated local interneurons is also compromised. Our data suggest that alterations in the growth of the DGNs do not contribute to the dramatic reduction in size of the olfactory neutrophils following serotonin depletion, but that cell proliferation and survival among the local interneurons are regulated by serotonin during development. Reduced numbers of local interneurons are therefore one likely reason for the growth reduction observed after serotonin depletion.

Serotonin depletion in vivo inhibits the branching of olfactory projection neurons in the lobster deutocerebrum.

Serotonin depletion during embryogenesis has been shown previously to retard the growth of the olfactory and accessory lobes of the lobster deutocerebrum (Benton et al., 1997). The present study was undertaken to determine whether morphological changes in the interneurons innervating these lobes contribute to this growth retardation. We examined the effects of in vivo serotonin depletion using 5,7-dihydroxytryptamine (5,7-DHT) on the morphology of the olfactory projection neurons, one of two major classes of interneurons that innervate both lobes. Intracellular dye fills of olfactory projection neurons in normal embryos showed that each neuron extensively innervates either the olfactory or accessory lobe before projecting to neuropil regions in the protocerebrum. In embryos injected with 5,7-DHT, however, the deutocerebral arbors of 13.5% of the olfactory projection neurons examined were either markedly reduced compared with normal neurons or absent. Affected neurons also exhibited a number of additional aberrant morphological features suggesting that these neurons represent cells that were affected during their initial morphogenesis. Olfactory projection neurons with aberrant morphologies were also encountered, although less frequently (7.5% of the neurons examined), in control (sham-injected) embryos indicating that the sham injections can affect the development of the brain. This observation provides insights into the nature of effects seen in control embryos in previous experiments (Benton et al., 1997). The results of the present study indicate that in vivo serotonin depletion inhibits the branching of olfactory projection neurons and suggest, therefore, that one of the functions of serotonin during normal development is to promote the ingrowth of these neurons into the deutocerebral neuropils.

Simultaneous irradiation for prostate cancer: intermediate results with modern techniques.

PURPOSE: In this study of men with early stage prostate cancer we evaluated treatment outcome after modern simultaneous irradiation, comprising transperineal implantation followed by external beam radiation. Disease-free survival rates were calculated according to an undetectable prostate specific antigen (PSA) nadir. MATERIALS AND METHODS: From 1992 to 1996, 689 men with clinical stage T1-T2, N0, Nx prostate cancer were treated with ultrasound guided transperineal 125iodine seed implantation followed 3 weeks later by external beam radiation. Disease-free status was defined as the achievement and maintenance of a PSA nadir of 0.2 ng./ml. or less. Median followup was 4 years (range 3 to 7). None of these men received neoadjuvant or adjuvant hormonal therapy. RESULTS: Overall 5-year disease-free survival was 88%. The 5-year rate according to PSA 4.0 ng./ml. or less, 4.1 to 10.0, 10.1 to 20.0 and greater than 20.0 was 94%, 93%, 75% and 69%, respectively. Multivariate analysis revealed that pretreatment PSA was the strongest indicator of subsequent disease-free status in regard to Gleason score or clinical stage. CONCLUSIONS: Intermediate treatment outcome analysis of modern simultaneous radiation supports the principles of radiation dose intensification for intracapsular disease plus the treatment of potential microscopic capsular penetration.

A new look at embryonic development of the visual system in decapod crustaceans: neuropil formation, neurogenesis, and apoptotic cell death.

In recent years, comparing the structure and development of the central nervous system in crustaceans has provided new insights into the phylogenetic relationships of arthropods. Furthermore, the structural evolution of the compound eyes and optic ganglia of adult arthropods has been discussed, but it was not possible to compare the ontogeny of arthropod visual systems, owing to the lack of data on species other than insects. In the present report, we studied the development of the crustacean visual system by examining neurogenesis, neuropil formation, and apoptotic cell death in embryos of the American lobster, Homarus americanus, the spider crab, Hyas araneus, and the caridean shrimp, Palaemonetes argentinus, and compare these processes with those found in insects. Our results on the patterns of stem cell proliferation provide evidence that in decapod crustaceans and hemimetabolous insects, there exist considerable similarities in the mechanisms by which accretion of the compound eyes and growth of the optic lobes is achieved, suggesting an evolutionary conservation of these mechanisms.

From embryo to adult: persistent neurogenesis and apoptotic cell death shape the lobster deutocerebrum.

Neuronal plasticity and synaptic remodeling play important roles during the development of the invertebrate nervous system. In addition, structural neuroplasticity as a result of long-term environmental changes, behavioral modifications, age, and experience have been demonstrated in the brains of sexually mature insects. In adult vertebrates, persistent neurogenesis is found in the granule cell layer of the mammalian hippocampus and the subventricular zone, as well as in the telencephalon of songbirds, indicating that persistent neurogenesis, which is presumably related to plasticity and learning, may be an integral part of the normal biology of the mature brain. In decapod crustaceans, persistent neurogenesis among olfactory projection neurons is a common principle that shapes the adult brain, indicating a remarkable degree of life-long structural plasticity. The present study closes a gap in our knowledge of this phenomenon by describing the continuous cell proliferation and gradual displacement of proliferation domains in the central olfactory pathway of the American lobster Homarus americanus from early embryonic through larval and juvenile stages into adult life. Neurogenesis in the deutocerebrum was examined by the in vivo incorporation of bromodeoxyuridine, and development and structural maturation of the deutocerebral neuropils were studied using immunohistochemistry against Drosophila synapsin. The role of apoptotic cell death in shaping the developing deutocerebrum was studied using the terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling method, combined with immunolabeling using an antiphospho histone H3 mitosis marker. Our results indicate that, in juvenile and adult lobsters, birth and death of olfactory interneurons occur in parallel, suggesting a turnover of these cells. When the persistent neurogenesis and concurrent death of interneurons in the central olfactory pathway of the crustacean brain are taken into account with the life-long turnover of olfactory receptor cells in crustacean antennules, a new, highly dynamic picture of olfaction in crustaceans emerges.

Serotonin depletion by 5,7-dihydroxytryptamine alters deutocerebral development in the lobster, Homarus americanus.

The olfactory and accessory lobes constitute prominent histological structures within the larval and mature lobster deutocerebrum, and both are associated with a dense innervation from paired serotonergic nerve cells, the dorsal giant neurons (DGNs). During development, the cell bodies of the DGNs are the first central somata to express serotonin (5-HT), and the onset of their 5-HT immunoreactivity coincides with the beginning of accessory lobe formation. In contrast, the olfactory lobe anlagen emerge much earlier and grow in the apparent absence of serotonin. The role of serotonergic input for the development of these brain structures was investigated in lobster embryos after serotonin had been depleted pharmacologically with the neurotoxin 5,7-dihydroxytryptamine. A approximately 90% reduction of serotonin was confirmed in eggs using high-performance liquid chromatography with electrochemical detection. Morphometric analyses suggested that serotonin depletion dramatically slowed the growth of olfactory and accessory lobes, although glomeruli differentiated at the normal time in both areas. The toxin exhibited a high degree of specificity for serotonergic neurons and associated target regions, and serotonin depletion persisted for at least 2 months following treatment. The goal of future experiments is to determine which of the cell types that innervate the olfactory and accessory lobes are affected by toxin treatment, thereby resulting in the retarded growth of these areas.

Amines and peptides in the brain of the American lobster: immunocytochemical localization patterns and implications for brain function.

The distributions of serotonin- (5HT-), substance P- (SP-), small cardioactive peptideb- (SCPb-), and histamine- (HA-) like immunoreactivities were examined in the adult lobster supraesophageal ganglion. Vibratome sections were labeled using avidin-biotin-peroxidase immunocytochemical methods. The localization patterns for each substance were assessed in 21 regions within the median protocerebrum, deutocerebrum, and tritocerebrum. Each immunoreactivity has a unique distribution within the brain; however, most regions are immunoreactive for more than one neurotransmitter. Of particular interest are SP-immunoreactive protocerebral neurons that contact olfactory projection neurons and appear homologous to those found in other crustaceans. Regional differences in immunolabeling within the deutocerebral olfactory and accessory lobes suggest that specific areas within individual olfactory lobe glomeruli serve distinct functions in olfactory processing, and that subpopulations of accessory lobe glomeruli are innervated by different groups of neurons. This detailed comparison of the labeling patterns also has allowed us to define the anatomical connectivity between several cell body clusters, fiber tracts, and neuropil areas in the lobster brain.

Glomerular organization in developing olfactory and accessory lobes of American lobsters: stabilization of numbers and increase in size after metamorphosis.

Olfactory glomeruli are columnar and radially arranged at the periphery of the primary chemosensory areas, the olfactory lobes (OLs), in the American lobster Homarus americanus. The number of olfactory glomeruli reaches nearly 100/lobe in midembryonic life, increases rapidly during larval life, and stabilizes at about 200 in juvenile and adult lobsters. The accessory lobes (ALs), higher order integration areas, are composed of cortical columns and of spherical glomeruli. Two populations of spherical glomeruli are defined, the cortical glomeruli located at the bases of the columns, and the medullary glomeruli scattered throughout the ALs. Both cortical columns and spherical glomeruli are seen for the first time in the second larval stage. There are about 1000 cortical columns and 1700 glomeruli/AL in the postlarva and these numbers remain constant during the life of the lobster. In both OLs and ALs, it is the size of the interglomerular spaces and of the glomeruli themselves that increases. Therefore, the data suggest that in both OLs and ALs the glomeruli were already generated when the lobster metamorphoses (stage III to IV) and switches from a planktonic to a benthic existence, and that the new sensory neurons that are formed at each molt in the antennulae grow into existing olfactory glomeruli. Stability of the glomerular population in the primary olfactory centers, once the full complement of glomeruli is acquired, has also been reported in insects, fish, and mammals.

Hemorrhagic colitis and pseudomelanosis coli in ipecac ingestion by proxy.

This report describes a toddler with chronic diarrhea, vomiting, and hypotonia due to surreptitious administration of syrup of ipecac by his mother (Munchausen's syndrome by proxy). Several features of this case distinguish it from previous reports of chronic ipecac ingestion in childhood: the development of grossly bloody stools; radiologic, endoscopic, and biopsy evidence of a chronic moderate colitis resembling ulcerative colitis; and the histologic finding of pseudomelanosis coli, providing an important clue to toxic ingestion. The significance and possible mechanism for genesis of pseudomelanosis coli is discussed. This case emphasizes the variability in presentation and difficulty in diagnosing long-term ipecac ingestion by proxy. Ipecac toxicity should be considered in children with unexplained colitis and vomiting.

Presynaptic serotonergic dysfunction in patients with Alzheimer's disease.

Indices of presynaptic serotonergic nerve endings were assayed in neocortical biopsy samples from patients with histologically verified Alzheimer's disease. The concentrations of 5-hydroxytryptamine (serotonin) and 5-hydroxyindoleacetic acid, serotonin uptake, and K+-stimulated release of endogenous serotonin were all found to be reduced below control values. Changes occurred in samples from both the frontal and temporal lobes, but they were most severe (at least a 55% reduction) in the temporal lobe. This is indicative of substantial serotonergic denervation. Values for serotonergic markers in Alzheimer's disease samples did not show correlations with rating of the severity of dementia, indices of cholinergic innervation, or senile plaque and cortical pyramidal neurone loss. However, neurofibrillary tangle count and an index of glucose oxidation (both probably reflecting pyramidal cells) correlated with the concentration of 5-hydroxyindoleacetic acid.

Choline acetyltransferase activity and histopathology of frontal neocortex from biopsies of demented patients.

Cortical biopsies were taken from the frontal lobe of 25 patients with presenile dementia. Choline acetyltransferase (ChAT) activity, and in some specimens the high affinity uptake of choline, was used to estimate loss of cholinergic nerve terminals. Of the 15 samples with varying degrees of histological evidence of Alzheimer's disease (AD) 14 were from clinically suspected examples of the disease. There was significant loss of ChAT in 10 of the 15 compared with control and the mean activity was also highly significantly reduced (to 41% of control). The deficit was found in patients examined within a year of onset of symptoms. In 6 biopsies from clinically suspected cases of AD without diagnostic histological features there was loss of activity in only one, subsequently shown to have Jakob-Creutzfeldt disease. The remaining samples were two of vascular dementia (no loss of ChAT), one probable disorder of white matter (no loss of activity) and one undiagnosed disorder (with loss of ChAT activity). Thus most patients without histologically demonstrated AD had no evidence of a presynaptic cholinergic defect. It was concluded that suspected cases of AD particularly suitable for putative cholinergic therapy were those with an onset of the disease at 55 to 65 and an absence of family history.